Our annotation of plasmids pMOL28 and pMOL30 confirmed all previously known heavy metal resistance clusters (24
), identified a few additional ORFs within these clusters, and described plasmid maintenance and transfer genes, as well as IS elements. However, the function of most of the newly described genes remains hypothetical or is unknown. Although these findings may suggest that these ORFs do not have any real function and therefore might be considered evolutionary remnants, microarray data has shed more light on their possible role because they are over- or underexpressed in the presence of heavy metals.
Plasmid pMOL28 shares a common set of genes with plasmids pSym of R. taiwanensis
and pHG1 of R. eutropha
H16 (Fig. ). This common set contains orthologs for basic plasmid functions in a ~90-kb backbone indicative of a common origin. This situation resembles the genesis of IncPβ plasmids, which also contain a conserved region involved in plasmid maintenance next to a variety of functional modules concerned with resistance to antibiotics, mercury, or xenobiotics or combinations thereof (8
). Plasmid pMOL30, in contrast, belongs to another plasmid family; parA
, and some other closely linked genes of pMOL30 are highly similar to B. vietnamiensis
Besides the determinants for metal resistance, other plasmid-borne genes were strongly induced by heavy metals, and some of these genes are involved in conjugative transfer (such as pMOL28 trbN
, and trbJ
), transposition (Tn4378
), and membrane maintenance. Of particular interest for the latter function are the putative glycosyltransferase genes that are situated between known metal resistance loci and that appear to be organized as they are in Shigella flexneri
). Their products may play a role in maintaining the integrity of the cell wall. Thus, the induction of the gtr
gene clusters in the presence of high concentrations of heavy metals suggests that such exposure may heighten the demands for membrane biogenesis and restoration of the outer membrane lipopolysaccharides.
Some truncated IS elements also were overexpressed during exposure to heavy metals. The fact that the metal-induced partial IS elements encode only the DNA-binding domain opens the possibility that their corresponding proteins could have evolved to serve another function unrelated to gene mobility but possibly related to regulation. Interestingly, other researchers suggested that some eukaryotic mobile genetic elements might have evolved in such a way (44
An intriguing feature of the microarray data was the multiple-metal responses exhibited by several genes belonging to the cop
, and pbr
loci. These genes inevitably were induced by the expected substrate and also by other metals. Most notably, mer
genes were activated in response to Cd(II) and Pb(II) and cop
genes were activated in response to Zn(II), Cd(II), and Ni(II), while the expression of the cnr
genes rose in response to Cu(II) and Cd(II), usually by the same order of magnitude irrespective of which metal was tested. These observations contrast with the more specific metal responses that previously were noted with gene fusions (biosensors) (6
). The responses of these gene fusions, made with pMOL28 and pMOL30 as various in vivo and in vitro constructions, mostly relied on bioluminescence. Indeed, they were elicited mostly by the metallic substrates of the corresponding resistance proteins (6
). This apparent contradiction may mainly reflect the differences in the timing of the responses to metal induction; thus, the luminescent responses of the gene fusions are observed only after a couple of hours, while the results for quantitative PCR or microarrays correspond to a 30-min pulse. Nonetheless, data reported for cnr
genes and cnr
) are consistent with the concept of a multiple-metal response [e.g., up-regulation of cnr
genes in the presence of Cu(II), although this metal is not a substrate of cnr
] and experimental detection after short exposure times (10 min) (32
), followed by a more specific response [up-regulation in the presence of Ni(II) or Co(II), which are the main substrates of cnr
] when cells are exposed to the metals for longer times (32
Thus, multiple-metal responses could be transient phases in global resistance to heavy metals, with an early stage of multimetallic up-regulation of the metal resistance genes followed by a more substrate-specific response directed towards a particular metal. These apparent different phases involved in the response and resistance to heavy metals need to be investigated further by detailed kinetic studies during exposure. Recently, such an analysis was performed with copper-induced Pseudomonas aeruginosa
). For now, we hypothesize that such layered multiple responses would mean that the heavy metal resistance genes are controlled by various regulatory pathways with different regulators, each responding to several metals. The genes for these regulatory circuits could be located on the two plasmids, but they could also be located on the larger replicons.
Alternatively, highly specific sigma factors may be involved. Currently, 11 sigma factors have been recognized in C. metallidurans CH34, half of which are up- or down-regulated in the presence of heavy metals; furthermore, deleting five of these factors caused a decrease in heavy metal resistance (D. Nies, personal communication). The only sigma factor encoded on a plasmid (pMOL28), cnrH, is induced in the presence of Cu(II), Cd(II), Co(II), or Ni(II) (D. Nies, personal communication; this study). The cellular defense mechanisms of C. metallidurans against heavy metals might encompass several stages, implying a response to various signals, some of which are distinct from the substrates of the detoxification genes, with the corresponding genes located on the various replicons.
In both plasmids, mobile genetic elements may have participated in acquiring genes involved in heavy metal resistance. In pMOL28, the metal resistance island is flanked by inactivated IS elements belonging to the IS3 family (IS1071).
In pMOL30, the metal resistance genes are grouped in two putative islands separated by a small 13-kb region that contains some tra genes that might have belonged to the pMOL30 backbone before the acquisition of the “islands” (Fig. ) (region 130 to 140 kb from parA). These islands, as well as the pMOL28 island, do not seem to be mobile, which might reflect ancient acquisition.
For all the heavy metals tested, microarrays showed that the genes most up-regulated were located on the two plasmids. But many chromosomal genes and genes on the megaplasmid also are up-regulated. It would be interesting to compare these responses with the microarray data reported previously for Escherichia coli
) and for P. aeruginosa
) after exposure to copper. These microarray data (and likely the data for the CH34 chromosome and the megaplasmid) give an overview of possible microbial responses to moderate to high concentrations of heavy metals. pMOL28 and pMOL30 gene expression data probably describe the bacterial reaction to the most acute viable metallic stress for mesophilic heterotrophs growing at neutral pH. Classic metal resistance genes are only part of the gene arsenal on which bacterial survival depends. We expect that the transcriptomic data from this study will be a stepping stone for additional research on the unknown and hypothetical genes in the form of proteomics, mutagenesis, and phenotypic analyses.